Model for UPS regulation of AChR assembly. (A) Model of the ER illustrating the concurrent processes of assembly (top) and ERAD (bottom) of unassembled AChR subunits. Normally, only between 20 and 40% of subunits assemble into AChRs, while those remaining are retrotranslocated and degraded rapidly by the UPS. Masking or presentation of a putative ‘degron' (indicated by ^*) on each subunit during folding/oligomerization in the ER determines an assembly or degradation fate, respectively. (B) Loss of proteasome activity results in a decrease of unassembled subunit ERAD and an increase in AChR formation. As subunit retrotranslocation requires proteasome activity, subunits remain integrated in the ER with some retaining the potential for oligomerization into AChRs.

Proteasome inhibitors increase surface AChR expression. (A) C₂C₁₂ myotubes and α₂βɛδ AChR-expressing B23ɛ cells incubated with proteasome inhibitors (LACT, NLVS, MG-115 and MG-132) for 4.5 h were bound with ¹²⁵I-Bgt to assess surface AChR expression. Values were normalized to ¹²⁵I-Bgt binding to untreated cells (P<0.005). (B) Surface AChR turnover rates inferred from ¹²⁵I-Bgt degradation and release into growth media containing vehicle (CTRL ), LACT (•) or NH₄Cl (▴). t_1/2 values were estimated from single exponential curve fits (CTRL: 17.9 h; LACT: 18.1 h; NH₄Cl: 47.7 h). The inset graph shows total ¹²⁵I-Bgt binding for CTRL and LACT-treated cells at 0 h. (C) Delivery of newly assembled AChRs to the cell surface as measured by ¹²⁵I-Bgt binding at designated time points during treatment with LACT. Data are shown for CTRL (○) and LACT (•) in fmol of receptor. All graphs represent mean and s.e.m. values (n=3).

Proteasome inhibitors increase assembly and delivery of AChRs to the cell surface. (A) Pulse–chase assay in myotubes with LACT and unlabeled Bgt. Radiolabeled AChRs inserted into the plasma membrane were immunoprecipitated as part of receptor–toxin complexes by anti-Bgt. Individual subunits were resolved by SDS–PAGE. (B) α subunit band intensities from (A) are quantified and shown for CTRL (○) and LACT (•). (C) The experiment described in (A, B) was repeated in triplicate for 0 and 4.5 h time points. The α and β subunit band intensities were quantified and are shown as the percentage of CTRL (n=3). (D) Time course of ¹²⁵I-Bgt binding to intracellular myotube AChRs in the presence of LACT. TX-100-soluble fractions were bound overnight with ¹²⁵I-Bgt and immunoprecipitated by mAb35. Data are presented as the amount of AChR (fmol, n=3). (E) Affinity purification of newly assembled AChRs by Bgt-Seph from myotubes pulse labeled and chased in the presence of LACT for 90 min (n=3). Band intensities for α and β were quantified and are shown as in (C).

Structure and pharmacology of AChRs assembled in the presence of LACT. Proteasome inhibitor treatment increased surface AChR expression in C₂C₁₂ myotubes. The inset graphs in panels A, C and D display increased surface AChR expression (in fmol) with LACT for each experiment (∼25–40% above CTRL). (A) Bgt affinity for surface AChRs measured by the rate of ¹²⁵I-Bgt release at 4°C, following 4.5 h LACT treatment of myotubes to surface AChR expression (with ¹²⁵I-Bgt included during the final 2 h). Expressed as the percentage of cell surface-bound ¹²⁵I-Bgt remaining (n=3), t_1/2 values were estimated to be 8.4 days (CTRL, ○) and 9.2 days (LACT, •) by single exponential curve fits. (B) Sucrose gradient sedimentation of surface AChRs expressed during 4.5 h of proteasome inhibitor treatment. Newly inserted surface AChRs were bound with ¹²⁵I-Bgt using the technique described in Figure 2C and separated on 5–20% sucrose gradients. The quantity of bound ¹²⁵I-Bgt in each fraction (total 18) was normalized to the peak fraction (11). Surface AChRs expressed in both LACT- (•) and NLVS- (▾) treated myotubes sedimented identically to CTRL (○) at a 9S peak (fractions 10–12) but had 26% (LACT) and 23% (NLVS) more ¹²⁵I-Bgt binding than CTRL. After incubation with LACT, the ligands (C) dTC and (D) carb were competed against the initial rate of 10 nM ¹²⁵I-Bgt binding. Graphs are plotted as mean and s.e.m. values (n=3). IC₅₀ values and Hill coefficients were calculated from curve fits using a three-parameter Hill equation.

Unassembled AChR subunits are degraded by ERAD. (A) Representative autoradiographs from pulse–chase assays of AChR subunit-expressing cell lines show degradation of each subunit in the absence (left) and presence (right) of LACT. (B) Composite degradation rates for unassembled AChR subunits. Subunit band intensities were quantified for CTRL (○) and LACT (•) and plotted as a percentage of the starting amount (time=0). Data are shown as the mean±s.e.m. of at least three experiments. t_1/2 values for CTRL (α: 77 min; β-HA: 42 min; γ-HA: 30 min; ɛ-HA: 57 min; δ: 57 min) and LACT (α:123 min; β-HA: >180 min; γ-HA: 120 min; ɛ-HA: 180 min; δ: 111 min) were determined from the composite data. (C) Western blot of α subunit-expressing cells treated for 4.5 h with vehicle (CTRL, lane 1), MG-115 (lane 2), or treated with MG-115 and denatured by SDS prior to immunoprecipitation (lane 3). tsA201 cells treated with vehicle (CTRL, lane 4) or MG-115 (lane 5). Samples were immunoprecipitated by mAb P22 and probed with anti-Ub (top) and anti-α (bottom). (D) Western blot of β-HA, ɛ-HA or δ subunits from stably expressing cells treated with MG-115 probed with anti-Ub (top) and subunit-specific antibodies (bottom), as above. (E) Anti-Ub Western blot of assembled and unassembled AChR subunits in myotubes treated for 4.5 h with LACT. Assembled AChRs and complexes were affinity purified from cell lysates by Bgt-Seph (lanes 1 and 2) while residual non-Bgt binding/unassembled subunits were collected from SDS-denatured secondary supernatants by immunoprecipitation with mAbP22 (α, lanes 4 and 5), mAb148 (β, lanes 6 and 7) or mAb88b (δ, lanes 8 and 9).